1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the discovery of a DNA fragmentation factor that triggers nuclear changes during apoptosis. Methods and compositions for making and using the same are disclosed.
2. Description of Related Art
Apoptosis, or programmed cell death, is executed through a "suicide" program that is built into all animal cells (reviewed by White, 1996; Wyllie, 1995). Cells undergoing apoptosis show distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation and formation of apoptotic bodies (Wyllie, 1980). The biochemical hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments (Wyllie et al., 1980). Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a family of cysteine proteases with specificity for aspartic acid residues, including CED-3 of C. elegans, CPP32/Yama/Apopain of humans, and DCP-1 of Drosophila (Yuan et al, 1993; Xue et al., 1996; Femandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995; Song, et al., 1997). Recently, these proteins have been designated as caspases (Alnemri et al., 1996).
The most intensively studied apoptotic caspase is caspase-3, previously called CPP32/Yama/Apopain (Femandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995). Caspase-3 normally exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically when cells are signaled to undergo apoptosis (Schlegel et al., 1996; Wang et al., 1996). Multiple apoptotic signals, including serum withdrawal, activation of Fas, treatment with granzyme B, ionizing radiation, and a variety of pharmacological agents, activate caspase-3 (Chinnaiyan et al., 1996; Darmon, et al., 1996; Datta et al., 1996, 1997; Erhardt and Cooper, 1996; Hasegawa et al., 1996; Jacobsen et al., 1996; Martin et al., 1996; Schlegel et al, 1996).
A caspase-3-specific tetrapeptide inhibitor, Ac-DEVD-CHO, can abolish the ability of cytosol from apoptotic cells to induce apoptosis in normal nuclei and block the initiation of the cellular apoptotic program m response to apoptotic stimuli (Nicholson et al., 1995; Dubrez, et al., 1996; Jacobsen et al., 1996). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, owing to a lack of apoptosis in the brain (Kuida et al., 1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari et al., 1996). These data indicate that caspase-3 is both necessary and sufficient to trigger apoptosis.
The identified substrates of caspase-3 include poly(ADP-ribose) polymerase (PARP) (Tewari et al., 1995; Nicholson et al., 1995), sterol-regulatory element binding proteins (SREBPs) (Wang et al., 1995; 1996), the U1 associated 70 kDa protein (Caciola-Rosen et al., 1996), D4-GDI (Na et al., 1996), huntingtin (Goldberg et al., 1996), and the DNA-dependent protein Kinase (Casciola-Rosen et al., 1996; Song et al., 1996). It is not known whether the cleavage of any of these substrates plays a causal role in apoptosis.
Given that apoptosis is tightly regulated and has been linked to pathways that are dysregulated in a variety of diseases including cancer, it is important to identify mechanisms by which to control this process.